Multi-formed collagenous biomaterial medical device

ABSTRACT

The invention involves a submucosa tissue that has the capability of being shape formed or shape configured. The submucosa involves a purified form of submucosa tissue. Optionally, the submucosa can be packaged in such a manner to permit sterility or maintain sterility of the submucosa.

STATEMENT OF RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.08/916,490, filed Aug. 22, 1997, now issued as U.S. Pat. No. 6,206,931,which claims the benefit of U.S. Provisional Application Serial No.60/024,542 filed Aug. 23, 1996 and of U.S. Provisional ApplicationSerial No. 60/024,693 filed, Sep. 6, 1996; this application also claimsthe benefit of U.S. Provisional Application Serial No. 60/110,407 filedDec. 1, 1998.

TECHNICAL FIELD

The invention generally relates to a medical device and in particular,to a medical device comprising a collagenous biomaterial.

BACKGROUND OF THE INVENTION

A naturally occurring biomaterial for medical implantation is moredesirable that a synthetic implant. Synthetic implants tend to causeadverse reactions in a patient, including thrombosis, immune responses,and potentially restenosis in vascular applications. Therefore, amedical implant that reduces or eliminates these problems is a technicaladvance.

Collagenous biomaterials are known to be used in medical applications asmedical devices. As a naturally occurring biomaterial, the implantproduces less complications than a synthetic implant. Collagen is usedas an abundant source of protein, and is most notably derived frombovine skin. Collagen forms a matrix that is useable as an implant.However, as a biomaterial, it does not have good manipulationproperties, unless treated in other ways. In addition, one problem withthese material is that collagen biomaterials also carry with themantigens which cause an immune response in the patient. Therefore, aproduct that behaves like collagen in vivo yet is highly manipulativeand elicits less to no negative immune response is a technicalachievement.

SUMMARY OF THE INVENTION

The foregoing problems are solved and a technical advance is achievedwith the present invention. A new biomaterial comprising the submucosaof a tissue was discovered to have greater benefit than using collagen.For example, the submucosa is shown to exhibit more remodeling,regrowth, and regeneration of tissue upon implant. It has been shownthat submucosal tissue is absorbed by the patient and thus the patientdoes not require post-implantation procedures to remove the implant. Thesubmucosal tissue has been shown to elicit favorable immune responsethat leads to an accommodation of the submucosal implant versus arejection based response. Therefore, to further improve the submucosaltissue's industrial utility, the applicants have discovered that thisutility can be achieved by improving the submucosa's purity and formingthe submucosal tissue into various forms. Such forms, include, but arenot limited to sheets, sponges, fluidized, etc. The present inventionrelates to a purified form of submucosal tissue that is treated in sucha manner as to confer some shape memory and shape configuration to theimplant. However, the submucosal tissues are not limited to implants andcan be formed to be used in topical applications as well, such as wounddressings or wound plugs.

In addition, the problem of maintaining the sterility of the medicaldevice is solved by including the medical device in a pouch or pluralityof pouches. The pouches can be at least one of a gas permeable, sealed,hermetically sealed, sterile UV protected, and multiple pouched. Inaddition, the foregoing problems are solved by including processes ofmaking the medical device.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows two aspects of the invention as the biomaterialmultilaminate sheet or in sponge forms.

FIG. 2A shows one aspect of the invention as the biomaterial in a doublepouched package, wherein the package is in an open configuration.

FIG. 2B shows one aspect of the invention as the biomaterial in a closeddouble pouched package.

DETAILED DESCRIPTION

In the discussions herein, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, the following definitions are provided.

Bioburden—refers to the number of living microorganisms, reported incolony-forming units (CFU), found on and/or in a given amount ofmaterial. Illustrative microorganisms include bacteria, fungi, and theirspores.

Disinfection—refers to a reduction in the bioburden of a material.

Sterile—refers to a condition wherein a material has a bioburden suchthat the probability of having one living microorganism (CFU) on and/orin a given section of the material is one in one-million or less.

Pyrogen—refers to a substance which produces febrile response afterintroduction into a host.

Endotoxin—refers to a particular pyrogen which is part of the cell wallof gram-negative bacteria. Endotoxins are continually shed from thebacteria and contaminate materials.

Purification—refers to the treatment of a material to remove one or morecontaminants which occur with the material, for instance contaminantswith which the material occurs in nature, and/or microorganisms orcomponents thereof occurring on the material. Illustratively, thecontaminants may be those known to cause toxicity, infectivity,pyrogenicity, irritation potential, reactivity, hemolytic activity,carcinogenicity and/or immunogenicity.

Biocompatibility—refers to the ability of a material to pass thebiocompatibility tests set forth in International Standards Organization(ISO) Standard No. 10993 and/or the U.S. Pharmacopeia (USP) 23 and/orthe U.S. Food and Drug Administration (FDA) blue book memorandum No.G95-1, entitled “Use of International Standard ISO-10993, BiologicalEvaluation of Medical Devices Part-1: Evaluation and Testing.”Typically, these tests assay as to a material's toxicity, infectivity,pyrogenicity, irritation potential, reactivity, hemolytic activity,carcinogenicity, and/or immunogenicity. A biocompatible structure ormaterial when introduced into a majority of patients will not cause anadverse reaction or response. In addition, it is contemplated thatbiocompatibility can be effected by other contaminants such as prions,surfactants, oligonucleotides, and other biocompatibility effectingagents or contaminants.

Contaminant—refers to an unwanted substance on, attached to, or within amaterial. This includes, but is not limited to: bioburden, endotoxins,processing agents such as antimicrobial agents, blood, blood components,viruses, DNA, RNA, spores, fragments of unwanted tissue layers, cellulardebris, and mucosa.

Tela submucosa—refers to a layer of collagen-containing connectivetissue occurring under the mucosa in most parts of the alimentary,respiratory, urinary, integumentary, and genital tracts of animals.

The invention is generally directed to a medical device, comprising acollagenous biomaterial (also referred to as collagen-based matrices,tissue mucosa, tissue submucosa, intestines, biomaterial) and is furtherdescribed in the non-limiting disclosure set forth below.

One such collagenous biomaterial includes tissue mucosa, which alsofurther includes a tissue submucosa, which further includes a smallintestine submucosa (SIS), also described herein as tela submucosa. Telasubmucosa is a multi-laminate structure, comprising the tunicasubmucosa, lamina muscularis mucosa, and the stratum compactum.Collagenous materials can also have biotropic agents comprising at leastone of a proteoglycan, glycosaminoglycan, and growth factor. The SIS canbe made using the techniques described in Cook et al., WIPO PublicationWO 98/22158, dated May 28, 1998, which is the published application ofPCT/US97/14855, the disclosure of which is set forth below.

One type of collagenous mucosa is tela submucosa, and as with manyanimal tissues, is generally aseptic in its natural state, provided thehuman or animal does not have an infection or disease. This isparticularly the case since the tela submucosa is an internal layerwithin the alimentary, integumentary, respiratory, urinary, and genitaltracts of animals. Accordingly, it is generally not exposed to bacteriaand other cellular debris such as the epithelium of the intestinaltract. One feature of the present invention is the discovery that bydisinfecting the source tissue for the tela submucosa prior todelamination, the aseptic state of the tela submucosa layer can bepreserved or substantially preserved, particularly if the delaminationprocess occurs under sterile conditions.

Other sources of mucosa exist. For example, the mucosa can also bederived from vertebrate liver tissue as described in WIPO Publication,WO 98/25637, based on PCT application PCT/US97/22727; from gastricmucosa as described in WIPO Publication, WO 98/26291, based on PCTapplication PCT/US97/22729; from stomach mucosa as described in WIPOPublication, WO 98/25636, based on PCT application PCT/US97/23010; orfrom urinary bladder mucosa as described in U.S. Pat. No. 5,554,389; thedisclosures of all are expressly incorporated herein.

In particular, it has been discovered that disinfecting the telasubmucosa source, followed by removal of a purified biomaterialincluding the tela submucosa, e.g. by delaminating the tela submucosafrom the tunica muscularis and the tunica mucosa, minimizes the exposureof the tela submucosa to bacteria and other contaminants. In turn, thisenables minimizing exposure of the isolated tela submucosa biomaterialto disinfectants or sterilants if desired, thus substantially preservingthe inherent biochemistry of the tela submucosa and many of the telasubmucosa's beneficial effects.

A tela submucosa implantable collagen biomaterial according to thepresent invention can, as indicated above, be obtained from thealimentary, respiratory, urinary, integumentary, or genital tracts ofanimals. Preferably, the tela submucosa tissues, which arecollagen-based and thus predominantly collagen, are derived from thealimentary tract of mammals, such as cows, sheep, dogs, and mostpreferably from the intestinal tract of pigs. A most preferred source ofwhole small intestine is harvested from mature adult pigs weighinggreater than about 450 pounds. Intestines harvested from healthy,non-diseased animals will contain blood vessels and blood supply withinthe intestinal tract, as well as various microbes such as E. colicontained within the lumen of the intestines. Therefore, disinfectingthe whole intestine prior to delamination of the tela submucosasubstantially removes these contaminants and provides a preferredimplantable tela submucosa tissue which is substantially free of bloodand blood components as well as any other microbial organisms, pyrogensor other pathogens that may be present. In effect, this procedure isbelieved to substantially preserve the inherent aseptic state of thetela submucosa, although it should be understood that it is not intendedthat the present invention be limited by any theory. All that isrequired or be met is that the biomaterial satisfies the above-mentionedcriteria.

It is also desirable that the collagenous biomaterial according to thepresent invention be substantially free of any antiviral agents or anyantimicrobial type agents which can affect the biochemistry of thebiomaterial and its efficacy upon implantation. In the past, one methodof treating such tissue material is to rinse the delaminated tissue insaline and soak it in an antimicrobial agent, for example, as disclosedin U.S. Pat. No. 4,956,178. While such techniques can optionally bepracticed with isolated collagenous mucosa or submucosa of the presentinvention, preferred processes according to the present invention avoidthe use of antimicrobial agents and the like which can not only affectthe biochemistry of the collagenous biomaterial but also can beunnecessarily introduced into the tissues of the patient.

As discussed above, it has been discovered that a highly pure form of animplantable tela submucosa collagen biomaterial can be obtained by firstdisinfecting a tela submucosa source prior to removing a purifiedcollagen biomaterial including the tela submucosa layer, e.g. bydelaminating the tela submucosa source. It has also been discovered thatcertain processing advantages as well as improved properties of theresultant tela submucosa layer are obtained by this process, includinggreater ease in removing attached tissues from the submucosa layer, anda characteristic, low contaminant profile.

Processes of the invention desirably involve first rinsing the telasubmucosa source one or more times with a solvent, suitably water. Therinsing step is followed by treatment with a disinfecting agent. Thedisinfecting agent is desirably an oxidizing agent. Preferreddisinfecting agents are peroxy compounds, preferably organic peroxycompounds, and more preferably peracids. Such disinfecting agents aredesirably used in a liquid medium, preferably a solution, having a pH ofabout 1.5 to about 10, more preferably a pH of about 2 to about 6, andmost preferably a pH of about 2 to about 4. In methods of the presentinvention, the disinfecting agent will generally be used underconditions and for a period of time which provide the recovery ofcharacteristic, purified submucosa matrices as described herein,preferably exhibiting a bioburden of essentially zero and/or essentialfreedom from pyrogens. In this regard, desirable processes of theinvention involve immersing the tissue source (e.g. by submersing orshowering) in a liquid medium containing the disinfecting agent for aperiod of at least about 5 minutes, typically in the range of about 5minutes to about 40 hours, and more typically in the range of about 0.5hours to about 5 hours.

A preferred peroxy disinfecting agent is hydrogen peroxide. Theconcentration of hydrogen peroxide can range from about 0.05% to 30% byvolume. More preferably the hydrogen peroxide concentration is fromabout 1% to 10% by volume and most preferably from about 2% to 5% byvolume. The solution can or can not be buffered to a pH from about 5 to9. More preferably the pH is from about 6 to 7.5. These concentrationscan be diluted in water or in an aqueous solution of about 2% to about30% by volume alcohol. Most preferably the alcohol is ethanol. Thesolution temperature can range from about 15 to 50° C. More preferablythe solution temperature is from about 20 to 40° C. Most preferably, thesolution temperature is from about 32 to 37° C. The exposure time canrange from about 10 to 400 minutes. Preferably, the exposure time isfrom about 120 to 240 minutes. More preferably, the exposure time isfrom 180 to 210 minutes.

A preferred organic peroxide disinfecting agent is perpropionic acid.The concentration of perpropionic acid can range from about 0.1% to 10%by volume. More preferably the perpropionic acid concentration is fromabout 0.1% to 1.0% by volume and most preferably from about 0.2% to 0.5%by volume. These concentrations of perpropionic acid can be diluted inwater or in an aqueous solution of about 2% to about 30% by volumealcohol. Most preferably the alcohol is ethanol. The tela submucosatissue source can be exposed to the organic peroxide solution forperiods from about 15 minutes to about 40 hours, and more typically inthe range of about 0.5 hours to about 8 hours. Other peroxy disinfectingagents are suitable for use as described in “Peroxygen Compounds”, S.Block, in Disinfection, Sterilization and Preservation, S. Block,Editor, 4th Edition, Philadelphia, Lea & Febiger, pp. 167-181, 1991; and“Disinfection with peroxygens”, M. G. C. Baldry and J. A. L. Fraser, inIndustrial Biocides, K. Payne, Editor, New York, John Wiley and Sons,pp. 91-116, 1988.

Another oxidizing disinfecting agent is chlorhexidine(1,6-di(4-chlorophenyldiguanido)hexane) in its digluconate form. Theconcentration of chlorhexidine digluconate can range from about 0.1% to15% by weight. More preferably, the chlorhexidine digluconateconcentration is from about 0.1% to 2% by weight and most preferablyfrom about 0.2% to 5% by weight. The solution can or can not be bufferedto a pH from about 5 to 8. More preferably the pH is from about 5.5 to7. These concentrations can be diluted in water or in an aqueoussolution of about 2% to about 20% by volume alcohol. Most preferably thealcohol is ethanol at a concentration of about 5% to 10%. The solutiontemperature can range from about 15 to 30° C. The exposure time canrange from about 10 to 400 minutes. More preferably the exposure time isfrom about 30 to 60 minutes. Other chlorine agents are described in“Chlorhexidine”, G. W. Denton, in Disinfection, Sterilization andPreservation, S. Block, Editor, 4th Edition, Philadelphia, Lea &Febiger, pp. 274-289, 1991.

In preferred preparative processes, a peracid or other disinfectingagent can be dissolved in a dilute aqueous alcohol solution, preferablywherein the alcohol has from 1 to about 6 carbon atoms, and wherein thealcohol can generally comprise from about 1% to about 30% by volume ofthe solution. More preferred alcohols for use in the invention areselected from the group consisting of ethanol, propanols and butanols.Ethanol is a preferred alcohol for these purposes.

When a peracid is used in the disinfection, it is preferably selectedfrom the group consisting of peracetic acid, perpropionic acid orperbenzoic acid. Peracetic acid is the most preferred disinfectingagent. The peracetic acid is preferably diluted into about a 2% to about10% by volume alcohol solution. The concentration of the peracetic acidcan range, for example, from about 0.05% by volume to about 1.0% byvolume. Most preferably the concentration of the peracetic acid is fromabout 0.1% to about 0.3% by volume. Hydrogen peroxide can also be usedas a disinfecting agent. Alternatively, or in addition, the telasubmucosa tissue source, e.g. from small intestine, can be disinfectedutilizing disinfecting agents such as glutaraldehyde, formalin and thelike, which are also known for their ability to introduce substantialcrosslinking into collagen matrices, in contrast to the action of otherdisinfecting agents such as peracids which can be used to disinfectwithout introducing such crosslinking. Additionally, the tela submucosasource can be treated with radiation, e.g., gamma radiation, forpurposes of disinfection.

Variations on the disinfection process can also include the following:

1. Intestine is treated with 0.2% peracetic acid, 5% ethanol solution ata ratio of 10:1 solution to intestine ratio by weight. Solution has a pHof 2.6. Solution and intestine are vigorously mixed for two hours.

2. Intestine is treated with 1% peracetic acid, 25% ethanol solution ata ration of 5:1 solution to intestine ratio by weight. Solution has a pHof 2. Solution and intestine are vigorously mixed for one hour.

3. Intestine is treated with 1% peracetic acid, 15% ethanol, and 10%hydrogen peroxide solution at a ratio of 5:1 solution to intestine ratioby weight. Solution and intestine are vigorously mixed for one hour.

4. Whole small intestine is rinsed four times with high purity water for15 minutes. The intestine is then subjected to 1.5 MRAD Electron Beamradiation.

5. Whole small intestine is rinsed four times with high purity water for15 minutes. Lengthwise along a conveyor belt, the intestine is subjectedto high-intensity pulsed light which disinfects the intestine.

Following the treatment as described above, the tela submucosa layer isdelaminated from its source, e.g., whole intestine, cow uterus and thelike. It has been found that by following thispost-disinfection-stripping procedure, it is easier to separate the telasubmucosa layer from the attached tissues, e.g. at least from attachedtunica muscularis tissue, as compared to stripping the tela submucosalayer prior to disinfection. Moreover it has been discovered that theresultant tela submucosa layer in its most preferred form exhibitssuperior histology, in that there is less attached tissue and debris onthe surface compared to a tela submucosa layer obtained by firstdelaminating the tela submucosa layer from its source and thendisinfecting the layer. Moreover, a more uniform tela submucosa tissuecan be obtained from this process, and a tela submucosa having the sameor similar physical and biochemical properties can be obtained moreconsistently from each separate processing run. Importantly, a highlypurified, substantially sterile tela submucosa is obtained by thisprocess. The stripping of the tela submucosa source is preferablycarried out by utilizing a disinfected or sterile casing machine, toproduce a tela submucosa which is substantially sterile and which hasbeen minimally processed. A suitable casing machine is the Model 3-U-400Stridhs Universal Machine for Hog Casing, commercially available fromthe AB Stridhs Maskiner, Götoborg, Sweden. Therefore, the measuredbioburden levels are minimal or substantially zero. Of course, othermeans for delaminating the tela submucosa source can be employed withoutdeparting from the present invention, including for example those meanswell known in the art, including delaminating by hand.

It has also been discovered that more preferred processes according tothe present invention, not only will eliminate or significantly reducecontaminants contained in the tela submucosa collagen biomaterial, butalso will produce a tissue which exhibits no substantial degradation ofphysical and mechanical properties, e.g., differential porosity (i.e.wherein one side of the submucosa layer has greater porosity than theother side), and good strength, for example burst strength. Also, it hasbeen discovered that more preferred processes do not affect thedifferential porosity of the tela submucosa collagen biomaterial, whichultimately affects the level of efficacy of this tissue implant. Forexample, the tissue is not necessarily treated with a crosslinking agentor a material that disrupts the porosity or inherent, native structureof the collagen biomaterial. Moreover, when hydrogen peroxide isemployed, the biomaterial as a whole has greater porosity as well as ahigher oxygen content. This helps to ensure the absence of contaminantse.g., endotoxins, pyrogens, and the like.

Preferred collagen-based matrices of the invention, preferablysubmucosa-containing matrices, are also characterized by the lowcontaminant levels set forth in Table 1 below, each contaminant leveltaken individually or in any combination with some or all of the otherdisclosed contaminant levels. The abbreviations in Table 1 are asfollows: CFU/g=colony forming units per gram; PFU/g=plaque forming unitsper gram; μg/mg=micrograms per milligram; ppm/kg=parts per million perkilogram; and EU/g=endotoxin units per gram.

TABLE 1 FIRST SECOND THIRD PREFERRED PREFERRED PREFERRED FEATURE LEVELLEVEL LEVEL ENDOTOXIN <12 EU/g <10 EU/g <5 EU/g BIOBURDEN <2 CFU/g <1CFU/g <0.5 CFU/g FUNGUS <2 CFU/g <1 CFU/g <0.5 CFU/g NUCLEIC ACID <10μg/mg <5 μg/mg <2 μg/mg VIRUS <500 PFU/g <50 PFU/g <5 PFU/g PROCESSING<100,000 <1,000 <100 AGENT ppm/kg ppm/kg ppm/kg

Even more preferred collagen-based matrices of the invention contain anendotoxin level of less than 1 EU/g, and most preferably less than 0.5EU/g.

Purified collagen-based matrices according to the present invention canbe processed in a number of ways, to provide collagenous matrices usefulboth in vitro and in vivo. For example, the submucosa can be configuredto provide tissue grafts useful in vascular applications, e.g., asgenerally described in U.S. Pat. No. 2,127,903 and 4,902,508.

The tela submucosa of the invention possesses mechanical propertieshighly desirable for tissue graft materials in vascular applications,including low porosity index, high compliance, and a high burststrength. One skilled in the art will appreciate that the preferredtissue graft material will be of low enough porosity to preventintraoperative hemorrhage and yet of high enough porosity to allowextension of a newly-developed vasa vasorum through the graft materialto nourish the neointimal and luminal surface.

Tela submucosa tissue of the present invention can also be processed toprovide fluidized compositions, for instance using techniques asdescribed in U.S. Pat. No. 5,275,826. In this regard, solutions orsuspensions of the tela submucosa can be prepared by comminuting and/ordigesting the tela submucosa with a protease (e.g. trypsin or pepsin),for a period of time sufficient to solubilize the tissue and formsubstantially homogeneous solution. The submucosa starting material isdesirably comminuted by tearing, cutting, grinding, shearing or thelike. Grinding the submucosa in a frozen or freeze-dried state isadvantageous, although good results can be obtained as well bysubjecting a suspension of pieces of the submucosa to treatment in ahigh speed blender and dewatering, if necessary, by centrifuging anddecanting excess waste. The comminuted tela submucosa can be dried, forexample freeze dried, to form a powder. Thereafter, if desired, thepowder can be hydrated, that is, combined with water or buffered salineand optionally other pharmaceutically acceptable excipients, to form afluid tissue graft composition, e.g. having a viscosity of about 2 toabout 300,000 cps at 25EC. The higher viscosity graft compositions canhave a gel or paste consistency.

Fluidized tela submucosa of this invention finds use as an injectableheterograft for tissues, for example, bone or soft tissues, in need ofrepair or augmentation most typically to correct trauma ordisease-induced tissue defects. The present fluidized submucosacompositions are also used advantageously as a filler for implantconstructs comprising, for example, one or more sheets of tela submucosaformed into sealed (sutured) pouches for use in cosmetic ortrauma-treating surgical procedures.

In one illustrative preparation, tela submucosa prepared as describedherein is reduced to small pieces (e.g. by cutting) which are charged toa flat bottom stainless steel container. Liquid nitrogen is introducedinto the container to freeze the specimens, which are then comminutedwhile in the frozen state to form a coarse tela submucosa powder. Suchprocessing can be carried out, for example, with a manual arbor presswith a cylindrical brass ingot placed on top of the frozen specimens.The ingot serves as an interface between the specimens and the arbor ofthe press. Liquid nitrogen can be added periodically to the telasubmucosa specimens to keep them frozen.

Other methods for comminuting tela submucosa specimens can be utilizedto produce a tela submucosa powder usable in accordance with the presentinvention. For example, tela submucosa specimens can be freeze-dried andthen ground using a manual arbor press or other grinding means.Alternatively, tela submucosa can be processed in a high shear blenderto produce, upon dewatering and drying, a tela submucosa powder.

Further grinding of the tela submucosa powder using a prechilled mortarand pestle can be used to produce a consistent, more finely dividedproduct. Again, liquid nitrogen is used as needed to maintain solidfrozen particles during final grinding. The powder can be easilyhydrated using, for example, buffered saline to produce a fluidizedtissue graft material of this invention at the desired viscosity.

To prepare another preferred fluidized material, a tela submucosa powdercan be sifted through a wire mesh, collected, and subjected toproteolytic digestion to form a substantially homogeneous solution. Forexample, the powder can be digested with 1 mg/ml of pepsin (SigmaChemical Co., St. Louis, Mo.) and 0.1 M acetic acid, adjusted to pH 2.5with HCl, over a 48 hour period at room temperature. After thistreatment, the reaction medium can be neutralized with sodium hydroxideto inactivate the peptic activity. The solubilized submucosa can then beconcentrated by salt precipitation of the solution and separated forfurther purification and/or freeze drying to form a protease-solubilizedintestinal submucosa in powder shape.

Fluidized tela submucosa compositions of this invention find wideapplication in tissue replacement, augmentation, and/or repair. Thefluidized submucosal compositions can be used to induce regrowth ofnatural connective tissue or bone in an area of an existent defect. Byinjecting an effective amount of a fluidized submucosa composition intothe locale of a tissue defect or a wound in need of healing, one canreadily take advantage of the biotropic properties of the telasubmucosa. Interestingly, fluidizing SIS by comminution or enzymaticdegradation does not result in any appreciable loss of biotropicactivities, as shown in U.S. Pat. No. 5,275,826.

It is also possible to shape large surface area constructs by combiningtwo or more tela submucosa segments of the invention, for instance usingtechniques as described in U.S. Pat. No. 2,127,903 and/or InternationalPublication No. WO 96/32146, dated Oct. 17, 1996, publishingInternational Application No. PCT/US96/04271, filed Apr. 5, 1996. Thus,a plurality of tela submucosa strips can be fused to one another, forexample by compressing overlapping areas of the strips under dehydratingconditions, to form an overall planar construct having a surface areagreater than that of any one planar surface of the individual stripsused to shape the construct. Shapes can be made by using sutures,staples, biocompatible adhesives such as collagen binding pastes, ordehydrating overlapping structures then heating the structure asdescribed in U.S. Pat. No. 3,562,820.

As described herein, the invention can take many shapes, such as coiled,helical, spring-like, randomized, branched, sheet-like, tubular,spherical, fragmented, fluidized, comminuted, liquefied, suspended,gel-like, injectable, powdered, ground, sheared, and solid materialshape.

The tela submucosa powder can be used alone, or in combination with oneor more additional bioactive agents such as physiologically compatibleminerals, growth factors, antibiotics, chemotherapeutic agents, antigen,antibodies, enzymes, and hormones. Preferably, the powder-form implantwill be compressed into a predetermined, three-dimensional shape, whichwill be implanted into the bone region and will substantially retain itsshape during replacement of the graft with endogenous tissues.

Tela submucosa of the invention can also be used as a cell growthsubstrate, illustratively in sheet, paste or gel shape in combinationwith nutrients which support the growth of the subject cells, e.g.eukaryotic cells such as endothelial, fibroblastic, fetal skin,osteosarcoma, and adenocarcinoma cells (see, e.g. InternationalPublication No. WO 96/24661 dated Aug. 15, 1996, publishingInternational Application No. PCT/US96/01842 filed Feb. 9, 1996). Incertain forms, the tela submucosa substrate composition will support theproliferation and/or differentiation of mammalian cells, including humancells.

The inventive tela submucosa can also serve as a collagenous biomaterialin compositions for producing transformed cells, (see, e.g.,International Publication No. WO 96/25179, dated Aug. 22, 1996,publishing International Application No. PCT/US96/02136 filed Feb. 16,1996; and International Publication No. WO 95/22611 dated Aug. 24, 1995,publishing International Application No. PCT/US95/02251 filed Feb. 21,1995). Such compositions for cell transformation will generally includepurified tela submucosa of the present invention, for example influidized or paste shape as described in U.S. Pat. No. 5,275,826, incombination with a recombinant vector (e.g. a plasmid) containing anucleic acid sequence with which in vitro or in vivo target cells are tobe genetically transformed. The cells targeted for transformation caninclude, for example, bone progenitor cells.

In order to promote a further understanding of the present invention andits features and advantages, the following specific Examples areprovided. It will be understood that these specific Examples areillustrative, and not limiting, of the present invention.

EXAMPLE 1

Thirty feet of whole intestine from a mature adult hog is rinsed withwater. This material is then treated in a 0.2% by volume peracetic acidin a 5% by volume aqueous ethanol solution for a period of two hourswith agitation. The tela submucosa layer is then delaminated in adisinfected casing machine from the whole intestine. The delaminatedtela submucosa is rinsed four (4) times with sterile water and testedfor impurities or contaminants such as endotoxins, microbial organisms,and pyrogens. The resultant tissue was found to have essentially zerobioburden level. The tela submucosa layer separated easily andconsistently from the whole intestine and was found to have minimaltissue debris on its surface.

EXAMPLE 2

A ten foot section of porcine whole intestine is washed with water.After rinsing, this section of tela submucosa intestinal collagen sourcematerial is treated for about two and a half hours in 0.2% peraceticacid by volume in a 5% by volume aqueous ethanol solution withagitation. Following the treatment with peracetic acid, the telasubmucosa layer is delaminated from the whole intestine. The resultanttela submucosa is then rinsed four (4) times with sterile water. Thebioburden was found to be essentially zero.

EXAMPLE 3

A small section of the tela submucosa intestinal collagen material wassubcutaneously implanted in a rat. Within 72 hours, significantangiogenesis was observed.

EXAMPLE 4

Two sections of small intestine are processed by differing methods. Thefirst section is rinsed in tap water, disinfected for 2 hours in a 5% byvolume aqueous ethanol solution comprising 0.2% by volume peraceticacid, pH approximately 2.6, delaminated to the tela submucosa, rinsed inpurified water, divided into two samples and rapidly frozen. The secondsection is rinsed in tap water, delaminated to the tela submucosa,rinsed in purified water, placed in a 10% neomycin sulfate solution for20 minutes (as described in U.S. Pat. No. 4,902,508), rinsed in purifiedwater, divided into two samples and rapidly frozen. The fourabove-prepared samples are tested for bioburden and endotoxin levels.The first two samples each have bioburdens of less than 0.1 CFU/g andendotoxin levels of less than 0.1 EU/g. The second two samples haverespective bioburdens of 1.7 CFU/g and 2.7 CFU/g and respectiveendotoxin levels of 23.9 EU/g and 15.7 EU/g.

EXAMPLE 5

Three sections of small intestine are processed by differing methods.The first is rinsed in tap water, disinfected for 2 hours in a 5% byvolume aqueous ethanol solution comprising 0.2% by volume peraceticacid, pH about 2.6, delaminated to the tela submucosa, rinsed inpurified water, and rapidly frozen. The second is rinsed in tap water,delaminated to the tela submucosa, rinsed in purified water, disinfectedaccording to the methods of Example 1 in U.S. Pat. No. 5,460,962(treatment for 40 hours in a 0.1% by volume aqueous solution ofperacetic acid, buffered to pH 7.2), and rapidly frozen. The third isrinsed in tap water, delaminated to the tela submucosa, rinsed inpurified water, disinfected according to the methods of Example 2 inU.S. Pat. No. 5,460,962 (treatment in 0.1% by volume peracetic acid inhigh salt solution, buffered to pH 7.2), and rapidly frozen. All threesamples were tested for endotoxins. The endotoxin levels were <0.14 EU/gfor the first sample, >24 EU/g for the second sample, and >28 EU/g forthe third sample.

EXAMPLE 6

Two sections of porcine small intestine were infected with 7×10⁶ plaqueforming units (PFU) of virus. Both were exposed to a 0.18% peraceticacid, 4.8% aqueous ethanol solution at a nine-to-one weight ratio ofsolution to material. A first sample was immersed in this solution for 5minutes; the second was immersed for 2 hours. The material processed for5 minutes exhibited 400 PFU per gram of material. The material processedfor 2 hours exhibited zero PFU per gram of material.

EXAMPLE 7

Purified tela submucosa, prepared as described herein, was tested todetermine its nucleic acid content. Four samples of material weighing 5mg each were subjected to DNA/RNA extraction as detailed in the DNA/RNAIsolation Kit by Amersham Lifescience Inc., Arlington Heights, Ill.Nucleic acid quantitation was performed by spectrophotometricdetermination of solution optical densities at 260 nm and 280 nm. Theaverage nucleic acid content was 1.9±0.2 mg per milligram of material.

Small intestinal submucosa, prepared as described by U.S. Pat. No.4,902,508, was tested to determine its nucleic acid content. Foursamples of material weighing 5 mg each were subjected to DNA/RNAextraction as detailed in the DNA/RNA Isolation Kit by Amersham. Nucleicacid quantitation was performed by spectrophotometric determination ofsolution optical densities at 260 nm and 280 nm. The average nucleicacid content was 2.4±0.2 mg per milligram of material.

EXAMPLE 8

Sections of tela submucosa prepared according to the methods describedherein were sent to an independent testing laboratory (NAmSA, Inc.,Northwood, Ohio) for biocompatibility testing as described in thestandard ISO 10993. The samples were tested for USP Acute SystemicToxicity, USP Intracutaneous Toxicity, Cytotoxicity, LAL Endotoxin,material-mediated Pyrogenicity, Direct Contact Hemolysis, and PrimarySkin Irritation. The samples passed all tests, indicating that thematerial is biocompatible.

EXAMPLE 9

Using the procedure set forth in U.S. Pat. No. 5,460,962, two sampleswere analyzed. The first Kemp sample indicated an endotoxin levelgreater than 24 endotoxin units per gram and the second Kemp sampleindicated an endotoxin level greater than 28 endotoxin units per gram.Thus, when using the procedure set forth in Kemp '962, the endotoxinlevels fall outside the biocompatibility levels.

EXAMPLE 10

Using the procedures set forth in U.S. Pat. Nos. 4,902,508 and 5,372,821issued to Badylak, the endotoxin level shown ranges as high as 23.9endotoxin units per gram per sample. This falls outside the permissiblerange and thus does not the meet the criteria of biocompatibility. Theinvention, prepared in the above prescribed manner of disinfection firstthen delamination, was observed to have an endotoxin level of less than12 endotoxin units per gram, and more particularly, reported anendotoxin level of less than 5 endotoxin units per gram. Thus, thematerial of the present invention is biocompatible as defined above.

EXAMPLE 11

With reference to FIGS. 2A and 2B, in preparing a lyophilized SIS sponge10, comminuted SIS (with a mean particle size of approximately 150 μm)was centrifuged in a Beckman TJ-6 centrifuge with a speed of 1550×g for15 minutes. The supernatant was poured off leaving a dough-likeconsistency of SIS remaining. The material was then poured into variouspolycarbonate molds and frozen at cold temperatures, preferably at −80°C. for at least 2 hours. The material was then vacuum-dried for 6 hoursin a lyophilizing system with the condenser at −70° C. at a vacuumpressure of less than 100 millitorr, preferably 15 millitorr.Optionally, the sponge 10 can then be sterilized using ethylene oxide.The resulting structure was an SIS sponge 10, having a creamyishyellow-white color.

In varying the above procedures, the following attributes were noticed.Varying the “g” force from 0-15000×g, centrifugation at the higher “g”results in an increased sponge 10 density, a decreased spongeabsorbency, and increased tensile strength. If the material weresterilized by radiation instead, for example using 10-25 kGy, noted wasa decrease in tensile strength. Using comminuted hydrated SIS with apossible fragment size of 25 to 3000 μm produced a more condensed,higher density sponge, whereas larger fragments produced a less denselarger pore sized sponge. Adding sugar molecules to the comminutedhydrated SIS resulted in a increased pore size sponge. Furthermore, theaddition of cross-linking agents, such as DANECOL, carbodiimide, orglutaraldehyde, caused an increased tensile strength that did not breakdown as easily upon the addition of water. In addition, moderating thepH of the suspension, from 2-10, preferably 3-5, demonstrated that ahigher pH will reduce the tensile strength of the sponge. Since theresulting sponge can take the shape of the mold in which it is placed,modifying the mold shape will modify the sponge shape.

The resulting sponge finds applicability, inter alia, in providinghemostasis in a large wound; fills a large defect; provides a threedimensional structure for cell culture or in vivo growth; provides aplug to stop bleeding; can be an injectable form; can be used for vesselembolization.

EXAMPLE 12

In accordance with the present invention, the lyophilized SIS wascompared to other forms of SIS to provide the following data:

Non Single De- De- 4-Layered layered watered watered 4-Layered Vacuum-Lyophilized SIS SIS Lyophilized Pressed Sheet sponge Sponge SheetSheet** Absorbency 5.3 5.6 4.2 3.1 1.0 Level (g water per g ofsample)*** Density .26 .16 .20 .26 .65 (g/cm³) Load at 882 161 587 45015931 Failure of 1 cm wide strip (gf) Stress at 7737 57 1792 11,169237,240 failure (gf/cm²) **The 4-layered sheet was made in accordancewith U.S. Pat. No. 5,711,969 issued to Patel, Hiles, Whitson, Cheng,Badylak, and Kokini; and assigned to Purdue Research Foundation andMethodist Hospital, all of Indiana, USA. The material used was made inaccordance with U.S. Pat. No. 4,902,508, issued to Badylak et al.***British Pharmacopoeia, 1988, vol. II, App. XX-L, pA226.

Notably, with reference to FIG. 1, the increasing number of lyophilizedsheets 15 used indicated a decrease in the absorbency level toapproximately 1.01 grams of water per gram of sample. Furthermore, byincreasing the amount of water centrifuged out, the density increased.

EXAMPLE 13

With reference to FIG. 1, to prepare lyophilized multilaminate sheets15, a sheet 20 of hydrated SIS was laid down on a non-stick surface,such as polytetrafluoroethylene (TEFLON(R)) and smoothed to remove anyripples or entrapments between the sheet 20 and the non-stick surface. Asecond sheet was placed over the first sheet and smoothed out in thesame fashion. The process was repeated until the preselected number ofsheets 20 were stacked forming the multilaminate sheet structure 15. Athin layer of high purity water was glazed over the top sheet of SIS andthe material was frozen at −80° C. for at least 2 hours. The materialwas then vacuum dried at a vacuum pressure less than 100 millitorr(preferably less than 15 millitorr) in a condenser operating at −70° C.for six hours. The dried sheets were then cut into a preselected shape,sealed in packaging, and terminally sterilized using ethylene oxide.

By modifying the process in the manner described in Example 11, thesheet may have different attributes. For example, it was noted thatadjusting the pH of the hydrated SIS to between 2-10, preferably 3-5,demonstrated an increased lamination strength. In addition, a tissueglue may be present between the sheets to increase lamination strength.

EXAMPLE 14

In making the SIS into various forms and lyophilizing the product, itwas noted that the porosity index of the SIS changed. A sheet ofhydrated SIS was attached to the bottom of a water column exerting ahydrostatic pressure of approximately 35 inches. The amount of watercollected via pass through after 30 seconds was measured. The experimentwas repeated using a single sheet of lyophilized SIS under the watercolumn. In comparing the hydrated form of a single sheet of SIS with thelyophilized form, the hydrated form had a mean of 9.59 grams of watercollected, with a standard deviation of 5.22. The lyophilized form had amean of 0.2 grams of water with a standard deviation of 0.13.

With reference to FIGS. 2A and 2B, as with the lyophilized product, theproduct can be packaged in packages that are, inter alia, gas permeable,hermetically sealed, sterile, sealed, UV protected, double pouched, orsome combination thereof. By UV barriered or UV protected, it is meantthat the packages or pouches are made of a material that substantiallyblocks the contents from UV radiation. For example, the package cancomprise the material disclosed in U.S. Pat. No. 5,489,022, which issuedon Feb. 06, 1996 to Sabin Corp. (Bloomington, Ind.), the disclosure ofwhich is entirely incorporated by reference herein. Preferably, theproduct is packaged in gas permeable double pouched packages that have aUV barrier to protect the internal product by placing the product in thepackage and sealing it. Gas permeable packages are desirable as itfacilitates terminal sterilization with ethylene oxide. A double pouchedpackage system 25 is desirable in that it permits the user to open theexterior pouch 30 and drop the inner pouch 35 on sterile surfaces.However, the packaging may include a plurality of pouches nested withineach other, such that there is a pouch within a pouch within a pouch,etc. with the biomaterial being in the inner or innermost pouch. Bysterile, it is meant that the package is sterilized and is of suchconstruction and material as to keep the contents of the package andinner pouches sterile. The package can also contain a buffered solution50 to keep the product wet. The solution can also contain thepharmocologic agents described above to permit long-term infiltration ofthe agent into the collagenous biomaterial product. The solution chosencan also include the characteristic that the final package can beterminally sterilized and thus sterilize the solution withoutdeleteriously changing the solution characteristics.

In another embodiment of the invention, the medical device productcomprises a submucosa of a warm blooded vertebrate made in the mannerdescribed above. The submucosa has an endotoxin level less than 12endotoxin units per gram of submucosa and the submucosa further havingan absorbency level of greater than 1.01 grams of water per gram ofsubmucosa. The collagenous biomaterial further made by the processcomprising the steps of sterilizing a source of submucosal tissue fromthe sources identified above; delaminating the source of submucosaltissue to expose the submucosa; freezing the submucosa in the mannersdescribed above; and drying the submucosa under vacuum pressure.Optionally, the collagenous biomaterial may be comminuted by grinding,shearing, or fragmenting the biomaterial in the manner described above.In addition, the collagenous biomaterial can further include packagingthe biomaterial in at least one of a gas permeable, sealed, hermeticallysealed, sterile, UV protected, and a plurality of pouches.

The collagen biomaterial can be made radiopaque by a variety ofconventional procedures, none of which has yet been applied to telasubmucosa. In one embodiment of the invention, the collagen material hasa shape, namely made into sheets, either in lyophilized ornon-lyophilized form. With reference to FIGS. 1, 2A, and 2B, anyradiopaque substance 40, including but not limited to, tantalum such astantalum powder, can be spread along the surface of the tela submucosa,such as on the serosal side. Other radiopaque materials 40 comprisebismuth and barium, including but not limited to, bismuth oxychlorideand barium sulphate, as well as other conventional markers. As usedherein, the term “disposed” on shall be construed to include disposedon, disposed throughout, disposed in, disposed with, disposed alongwith, applied on, applied with, applied through, applied in, applied inconjunction with, and the like. With particular reference to telasubmucosa, the differential porosity of the material can enable moreradiopaque material 40 to be disposed on the tela submucosa.

In one particular embodiment, radiopaque marker tantalum powder wasdisposed on a sheet of tela submucosa by rubbing it onto the serosalside of the tela submucosa. The tela submucosa was then made intovarious shapes, such as, but not limited to, having the shape of abrush-like, braided, branched, helical, spherical, cubic, cylindrical,tubular, injectable, randomized, layered, and sheet-like shapes. Forexample, an injectable shape of the invention can be readily made bycomminuting the invention into small fibrils, fragments, or the like,then suspending them in solution, such as, but not limited to, abiocompatible gelatin suspension. Due to the viscosity of the gelatinsuspension, the invention, when injected into the lumen of an aneurysm,will stay in the lumen and provide the therapeutic benefit to theaneurysm.

The invention and collagenous biomaterial can be made in layers. In thismanner, the collagenous material can increase its structural integrity,strength, and applicability. In one embodiment of the invention, a duallayer of collagenous biomaterial can be used in sheets and either theradiomarker or pharmacologic agent, or both can be disposed in betweenthe layers.

In similar fashion, the pharmacologic agent 45 can be disposed on thecollagenous material. As used herein, the pharmacologic agent 45includes, but is not limited to, growth factors, proteins,proteoglycans, glycosaminoglycans, physiological compatible minerals,antibiotics, chemotherapeutic agents, enzymes, drugs, and hormones. Theagent can be disposed on the serosal or mucosal sides of the submucosa,or can be disposed on the same or opposite sides of the collagenousmaterial. Consideration of placement can be important depending on thedesired shape of the final device. For example, where the shape istubular, it can be desirable to place the pharmacologic agent 45 on theabluminal surface since that surface will be in contact with thesurrounding tissue. On the other hand, if systemic release of the agent45 is contemplated, then the agent can be placed on the lumenal side topermit the blood to contact the agent and carry it away. Utilizing thebraid shape, each individual strip can be treated with different agents.

It will be appreciated that variations of the above-described processingprocedures are intended to be within the scope of this invention. Forexample, the source tissue for the collagenous biomaterial, e.g.,stomach, whole intestine, cow uterus and the like, can be partiallydelaminated, treated with a disinfecting or sterilizing agent followedby complete delamination of the tela submucosa. Illustratively, attachedmesentery layers, and/or serosa layers of whole intestine can beadvantageously removed prior to treatment with the disinfecting agent,followed by delamination of remaining attached tissues from the telasubmucosa These steps can or can not be followed by additionaldisinfection steps, e.g., enzymatic purification and/or nucleic acidremoval. Alternatively, the tela submucosa source can be minimallytreated with a disinfecting or other such agent, the tela submucosadelaminated from the tunica muscularis and tunica mucosa, followed by acomplete disinfection treatment to attain the desired contaminantlevel(s). All such variations and modifications are contemplated to be apart of the process described herein and to be within the scope of theinvention.

Many alterations and modifications can be made by those of ordinaryskill in the art without departing from the spirit and scope of theinvention. The illustrated embodiments have been shown only for purposesof clarity and examples, and should not be taken as limiting theinvention as defined by the appended claims, which include allequivalents, whether now, or later devised.

What is claimed is:
 1. A collagenous biomaterial medical device,comprising: a laminate having a plurality of layers of lyophilizedsubmucosa; said submucosa having an absorbency level of greater than1.01 grams of water per gram of submucosa and a density between about0.10 and about 0.40 grams per cubic centimeter.
 2. The collagenousbiomaterial medical device of claim 1, wherein the submucosa has anabsorbency level of greater than 2.0 grams of water per gram ofsubmucosa.
 3. The collagenous biomaterial medical device of claim 2,wherein the submucosa is a member selected from the group consisting ofsmall intestinal submucosa, stomach submucosa and urinary bladdersubmucosa.
 4. The collagenous biomaterial medical device of claim 3,wherein the submucosa is small intestinal submucosa.
 5. A collagenousbiomaterial medical device, said collagenous biomaterial medical devicecomprising a laminate structure including submucosa having an absorbencylevel of greater than 1.01 grams of water per gram and a density betweenabout 0.10 and about 0.40 grams per cubic centimeter, said laminatestructure prepared by a process comprising: providing a plurality ofsubmucosa layers in contact with one another; lyophilizing the pluralityof submucosa layers in contact with one another.
 6. The collagenousbiomaterial medical device of claim 5, wherein the submucosa has anabsorbency level of greater than about 2 grams of water per gram.
 7. Thecollagenous biomaterial medical device of claim 6, wherein the whereinthe submucosa is a member selected from the group consisting of smallintestinal submucosa, stomach submucosa and urinary bladder submucosa.8. The collagenous biomaterial medical device of claim 7, wherein thesubmucosa is small intestinal submucosa.